PMID25869666

[manulera]

PMID25869666

Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways

Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors.

Results

Figure 1

• [x] Delayed nuclear congression
  • [x] klp2 -> 20 min delay
  • [x] dhc1 -> 5 min delay
  • [x] tea2 -> 13 min delay
• [x] Abolished nuclear congression:
  • [x] dhc1 klp2 -> 2 of 87-> penetrance high ()

Figure S1

• [x] Delayed nuclear fusion once they have congressed
  • [x] dhc1, S1D

Figure 2

Figure 3

Figure 4

• [x] Delayed nuclear congression
  • [x] dli1D
  • [x] dhc1D(1-1266)
• [x] Abolished nuclear congression (79% of cells)
  • [x] dli1D klp2
• [x] Abolished nuclear contression (100% cells)
  • [x] dhc1D(1-1266) klp2D
• [x] Possible NOT annotation:
  • [x] ssm4D klp2D still congression (dynactin is not necessary)
  • [x] ssm4D still recruits dhc1 to the SPB
  • [x] same observations for dic1 (connects dynein and dynactin) (Fig. S4)
• [x] Abolished dynein localization from SPB (Before fusion never, only 54% after fusion):
  • [x] dl1D
• [x] Very minor rescue of klp2D by OE of dhc1 (from 48 to 40 total time).

Figure 5

• [x] Expression of dhc1 from nmt41 during interphase -> 10% localization to SPB with and without dl1
• [x] dl1 alone when overexpressed without dhc1 ectopic expression is not recruited to the SPB.
• [x] Co-expression of both dhc1 and dl1 from nmt41 brings recruitment to 90%.
• [x] ectopic expression of dli1 during interphase increases cellular levels of dhc1 as seen by western blot
• [ ]

FYPO

• [x] FYPO:0006129. Make the term more specific. Create an issue.

GO

• [x] dhc1 -> nuclear congression

• [x] klp2 -> nuclear congression

• [x] Dli1?

• [x] Dhc1 data could be used for mins end directed IMP for GO.

• [x] klp2 localization at plus ends during karyogamy. still conditioned by Mal3. (Fig. 3)

• [x] Klp2 recruited to plus ends by Mal3 (Fig. S3A)

• [x] Dhc1 recruited to SPB during karyogamy (89% of cells), Fig. 3B

  • [x] Yamamoto et al. 1999 has shown that during horsetail dhc1 is in the cortex.
  • [ ]

    Existing annotations.

• [x] GO term of karyogamy from pkl1.

  • This paper shows more direct evidence that pkl1 does not change karyogamy. klp2D and dhc1D always congress, but not the double mutant. In addition pkl1D klp2D is as bad as pkl1D
  • In particular, 44% of the asci from klp2D,dhc1-d1D klp2D,dhc1-d1D crosses and 52% from crosses of the triple delete strain contained more than four spores per ascus, suggesting that meiosis had proceeded in the absence of karyogamy to result in catastrophic rates of spore death (Figure 4A).

• [x] GO term of plus-end for klp2.

  • In Janson's paper this is not mentioned.

• [x] update cut7 minus AND plus end reference for this one: https://www.pnas.org/doi/full/10.1073/pnas.1611581113

  • [x] Crosslinking activity should also be added. GO ticket pending

• [x] Tea2 plus-end directed direct evidence: Bieling et al. 2007 speckles in Fig. 4A.

• [x] Include this as support for the GO terms, because it's better direct evidence than the existing annotion from Troxell.

Discuss with Val

• [x] GO? for dl1 since localization is co-dependent? Strong evidence from co-expression during interphase from nmt1 promoter (Fig. 5C)

[manulera]

• [x] This is waiting for https://github.com/pombase/fypo/issues/4139

[manulera]

• [ ] Annotate location dependency of klp2 on mal3 here and others, see #14